This proposal seeks to extend research on the regulation of the intrinsic blood coagulation pathway. Since hereditary protein S deficiency is associated with both venous and arterial thrombotic disease, protein S is an important physiologic antithrombotic factor. Research will focus on the protein S-dependent downregulation of the prothrombinase complex of the intrinsic coagulation pathway. We hypothesize that: i) the anticoagulant activities of protein S depend on its direct interactions with factors Va and Xa and on its direct association with APC; ii) specific residues on the surface of protein S are essential for binding of protein S to factor Va, factor Xa or to C4BP; and iii) similarly, residues on the surface of factor Va are essential for its binding to protein S, prothrombin and factor Xa. Preliminary work shows that the synthetic EGF- 1 module of protein S is a potent inhibitor of the APC cofactor anticoagulant activity of protein S and demonstrates the feasibility of proposed studies using synthetic protein S modules as competitive inhibitors of protein-protein interactions. We hypothesize that protein S can exist in isoforms that differ markedly in their APC cofactor and APC-independent anticoagulant activities. Based on our preliminary data, it is hypothesized that cardiolipin (CL) enhances the protein C pathway by increasing the activities of both APC and protein S and that the protein C pathway may be impaired in subjects with anti-CL antibodies who are at increased risk for thrombosis. Thus, the five specific aims include the following (1) To identify functionally essential surface regions and amino acid residues of protein S that are responsible for its interactions with factors Va and Xa. Structure-function studies will employ synthetic polypeptides, including individual EGF-like modules, the thrombin- sensitive loop, the gla domain, and chemically-ligated polypeptides containing various combinations of these domains. Amino acid substitutions in synthetic protein S modules will be used to identify functionally essential amino acids; (2) To purify, separate and characterize the various isoforms of protein S that differ in their APC- independent and APC cofactor anticoagulant activities; (3) To identify molecular regions on factor Va that are responsible for its interactions with factor Xa, prothrombin, and protein S. Kinetic and binding studies will employ synthetic peptides and heavy and light chains of factor Va as inhibitors of protein-protein interactions to identify essential regions and residues; (4) To identify regions on the surfaces of C4BP and protein S that are responsible for their complexation; and (5) To characterize the mechanisms by which cardiolipin (CL) and phosphatidylethanolamine (PE) enhance the anticoagulant activities of the protein C pathway. Binding studies will quantify whether CL or PE alters the affinities of protein S, APC, or factors Xa or Va for phospholipid vesicles or alters the interactions among these proteins. The proposed structure-function studies of protein S and of factor Va will contribute to our understanding of the pathophysiology of thrombosis and thereby eventually to the improved treatment of patients with thrombosis.